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Article

Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo

by
Leonid M. Irenge
1,2,
Jérôme Ambroise
1,
Bertrand Bearzatto
1,
Jean-François Durant
1,
Maxime Bonjean
1 and
Jean-Luc Gala
1,*
1
Center for Applied Molecular Technologies, Institute of Experimental and Clinical Research, Université Catholique de Louvain (UCLouvain), 1200 Woluwe-Saint-Lambert, Belgium
2
Defence Laboratories Department, ACOS Ops & Trg, Belgian Armed Forces, 1800 Peutie, Belgium
*
Author to whom correspondence should be addressed.
Microorganisms 2023, 11(2), 525; https://doi.org/10.3390/microorganisms11020525
Submission received: 28 January 2023 / Revised: 15 February 2023 / Accepted: 16 February 2023 / Published: 18 February 2023
(This article belongs to the Special Issue Advances in Antibiotic and Drug-Resistance Mechanisms)
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Abstract

:
Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.

1. Introduction

Infections caused by MDR Gram-negative bacteria (GNB) are becoming a major public health issue all over the world. Because of the limited antimicrobial treatment options, these infections are frequently associated with high morbidity, prolonged hospitalization, high healthcare costs, and poor outcome [1]. These bacteria include MDR ESBL-producing Enterobacteriaceae, which have been identified as a major contributor to the current AMR crisis by the World Health Organization (WHO) [2]. According to data from Asia, Latin America, Europe, and Africa, the dramatic increase in third-generation cephalosporin resistance in Enterobacteriaceae is mainly due to the spread of blaCTX-M-15 gene ESBL [3,4,5], with blaCTX-M-15 gene being the most common ESBL gene worldwide [6,7]. While most studies of MDR ESBL-producing Enterobacteriaceae have focused on Escherichia coli [8,9,10,11,12,13,14,15], few studies have characterized the whole genome of MDR ESBL-producing Klebsiella pneumoniae in Africa, even though this agent is one of the most widespread MDR ESBL-producing Enterobacteriaceae associated with extra-intestinal infections both in hospital and community settings [16,17,18,19,20,21]. Moreover, several strains of K. pneumoniae have been linked to multinational epidemics all over the world [21,22]. In the DRC, there is little epidemiological data on K. pneumoniae and its role in the emergence of hospital and community-acquired infections. Nonetheless, the few available studies that have been conducted in the country show an increasing prevalence of MDR K. pneumoniae in hospital-acquired infections [16,17,23]. We previously reported a high prevalence of MDR ESBL-producing E. coli, K. pneumoniae, and Enterobacter cloacae in the urinary tract and bloodstream infections in eastern DRC [16,17]. WGS performed later on MDR ESBL-producing ExPEC isolates from the same collection showed the high prevalence of the ST131 and ST405 pandemic clones carrying the plasmid-mediated CTX-M-15 gene as the primary cause of ESBL-production in EXPEC [9]. Accordingly, the purpose of this study was to characterize extra-intestinal EBSL-producing K. pneumoniae isolates from the South-Kivu province of DRC using WGS. These isolates were collected in the years 2013 through 2017. The WGS data allowed phylogenomic analyses, as well as the characterization of the virulome and the resistome.

2. Materials and Methods

2.1. Ethical Clearance

The study protocols were approved by the Ethical Review Board (ERB) of the Université Catholique de Bukavu, DRC (UCB/CIES/NC/001/2023). The ERB granted a waiver of informed consent after deciding that the study complied with international guidelines on antibiotic surveillance studies. Samples were analysed anonymously to protect confidentiality.

2.2. Bacterial Strains

Between September 2013 and June 2017, GNB (n = 2051) were isolated from patients who presented to the 385-bed General Hospital of Bukavu (South Kivu province, DRC) with urinary tract infections (n = 1608), bloodstream infections (n = 416), as well as peritonitis (n = 12), pleurisy (n = 7), and soft tissue infections (n = 8). GNB bacteria were isolated and identified at the species level in urine samples as previously described [16]. Likewise, GNB associated with bloodstream, peritoneal, pleural, and soft skin tissue infections were isolated and identified at the species level following the protocol established in 2014 at HGPRB [17]. All the K. pneumoniae isolates (n = 449) from this 2013–2017 collection represented 21.9% of all extra-intestinal GNB isolates. They were selected for additional analyses, including antimicrobial susceptibility testing.

2.3. Antimicrobial Susceptibility Testing

K. pneumoniae isolates were tested for susceptibility to 14 antimicrobial agents (i.e., amikacin, gentamicin, ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, imipenem, meropenem, nitrofurantoin, and colistin) by the method of minimum inhibitory concentration (MIC). The MIC for each of the 14 antimicrobial agents minus colistin was determined after 16–20 h incubation on Mueller–Hinton plates inoculated with suspensions of isolates at a fixed density (0.5 to 0.6 McFarland standard), using MIC test strips (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer’s recommendations. MIC for colistin was determined by the broth microdilution method, using the ComASP® Colistin 0.25–16 µg/mL panel (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer’s recommendations. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, which were updated in January 2022, were used to interpret the susceptibility or resistance status of each isolate. ESBL-production assay was performed by using the MIC Test Strip ESBL Ceftazidime (CAZ)/Ceftazidime + Clavulanic acid (CAL) panel (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer’s recommendations). E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains were used as ESBL-negative and positive controls, respectively, while E. coli ATCC 25922 and E. coli NCTC 13846 were used as colistin-susceptible and resistant strains, respectively.

2.4. Bacterial DNA Extraction

K. pneumoniae isolates were cultured overnight in 10 mL Luria-Bertani broth. Genomic DNA was isolated using the EZ1 Advanced XL Biorobot and the tissue DNA kit (Qiagen, Hilden, Germany) with the Bacterial card, according to the manufacturer’s instructions. For each isolate, genomic DNA was quantified using Qubit® fluorometric quantitation (ThermoFisher Scientific, Hillsboro, OR, USA) and normalized to 0.2 ng/μL.

2.5. Library Preparation and Next Generation Sequencing

The library was prepared using the Nextera XT DNA Library preparation kit (Illumina, San Diego, CA, USA) as previously described [12]. Briefly, bacterial DNA was fragmented, and then tagged with sequencing adapters using the Nextera transposome (Nextera XT DNA Library Preparation Kit, Illumina, San Diego, CA, USA). PCR reaction of 12 cycles was performed to amplify the DNA fragments and to incorporate indices for dual-indexed sequencing of pooled libraries. The 1 nM pooled library was denaturated and diluted prior to loading on a MiSeq paired-end 2 × 150 (MiSeq reagent kit V2 (300 cycles) or 2 × 300 base pairs (bp) (MiSeq reagent kit V3 (600 cycles) sequence run.

2.6. Genome Assembly, Annotation, and Alignment

Raw sequencing data from all K. pneumoniae isolates were submitted to the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena; submitted on 22 December 2022) and are available under accession number PRJEB56212. These data were analysed using the following bioinformatics pipeline. All reads were quality checked using FastQC v.0.11.9 [24], and processed using trimmomatic v.0.39 [25]. Paired-end reads from each K. pneumoniae isolate were assembled de novo using the Spades v.3.13.0 algorithm [26] to generate a draft genome sequence for each isolate. Quality assessment of the genome assemblies was performed using QUAST 5.0.2 [27]. In addition, the quality and completeness of the genome assemblies were confirmed using the BUSCO v.4.1.1 algorithm and the bacteria_odb10 dataset [28]
The PATRIC database [29] was used to identify complete or draft genomes of other K. pneumoniae strains isolated from patients in other sub-Saharan African countries. In-silico MLST typing was performed on all genomes by using the screen-Blast-mlst function of the Pathogenomics home-made R package (https://github.com/UCL-CTMA/Pathogenomics) and the K. pneumoniae allele sequences and MLST profiles available in the BIGSdb-Pasteur platform (https://bigsdb.pasteur.fr/klebsiella; accessed on 22 April 2022). It should be noted that the assignment of an allele type requires a perfect match with the corresponding sequence (i.e., 100% sequence identity and coverage).
All genomes were submitted to kSNP3.0 [30] using the default settings, with the exception of a k-mer size of 19 and the ‘-core’ option. This software identified SNPs without the need for genome alignment or a reference genome. The phylogenomic tree based on the core genome was visualized using the R package ggtree [31]. The Pathogenomics custom R package was also used to screen all genomes for the virulence factor genes described in the VFDB database (http://www.mgc.ac.cn/VFs/; accessed on 24 August 2022 ) [32] and which are specifically associated to K. pneumoniae. The thresholds for sequence identity and coverage were respectively set at 80% and 50%. The R package ggtree was used to display the coverage percentage of each virulence gene in grayscale heatmaps. In silico K-typing and O-typing were performed on draft genomes of the 37 K. pneumoniae isolates, using Kleborate (v.2.2.0) [33] and Kaptive (v.2.0.3) [34]. Each isolate was assigned a virulence score based on the presence or absence of the yersiniabactin (ybt), colibactin (clb), and aerobactin (iuc) genes, as previously described [33].
Simultaneously, each draft genome was screened for the presence of AMR genes and point mutations using the AMRFinderPlus 3.10.5 [35] tool developed by the National Center for Biotechnology Information (NCBI). The draft genomes were screened for the presence of plasmid incompatibility groups, in particular the IncF group. The Replicon Scheme Typing (RST) developed by Villa et al. [36] was used for classification of IncF plasmids according to the FAB (FII, FIA, FIB) formula. The DNA sequence of the novel FIA replicon was submitted to the curator of the pMLST database (https://pubmlst.org/plasmid/; accessed on 26 July 2022) for ST assignment.

3. Results

3.1. Bacterial Strains

A set of MDR ESBL-producing K. pneumoniae isolates were identified from the total K. pneumoniae (n = 89/449, ~20%) isolated from the GNB collection assembled between 2013 and 2017 at the Bukavu General Hospital, South Kivu, DRC. Due to isolate loss, either because of a problem with isolate storage or other technical issues, only 37 of the original 89 MDR ESBL-producing K. pneumoniae isolates were thoroughly analysed. The isolates were not duplicate clinical samples, but rather one sample per patient. The majority (n = 31, 83.8%) of the isolates were cultured from samples obtained from patients who were hospitalized and included all isolates from bloodstream (n = 19, 51.4%), urinary tract (n = 9, 24.3%), peritoneal fluid (n = 2, 5.4%), and soft tissue (n = 1, 2.7%). The remaining (16.2%) isolates (n = 6) were collected from patients with community-acquired urinary tract infections.

3.2. Antimicrobial Susceptibility Testing

All K. pneumoniae isolates (n = 37) were susceptible to imipenem, meropenem, and colistin while showing reduced susceptibility to ampicillin, amoxicillin, cefotaxime, ceftazidime, and ceftriaxone. While the majority of them (30 out of 37) showed reduced susceptibility to cefepime, they all exhibited the ESBL-production phenotype (Table 1 and Table 2).

3.3. Phylogenomic Analysis

SNP-based phylogenetic analysis of the MDR ESBL-producing K. pneumoniae from DRC revealed a high level of genetic diversity. The 37 K. pneumoniae isolates were distributed in 16 different clusters (Figure 1), each of which corresponded to a unique MLST profile (Table 3).
The most common sequence types (STs) were ST607 (n =6), ST48 (n = 6), ST340 (n = 5) and ST39 (n = 4). Other ST (i.e., ST15, ST874, ST1777 and ST37) had 2 isolates, while ST14, ST16, ST502, ST337, ST967, ST11, ST309 and ST2094 had only one. ST48 isolates (n = 6) were all from blood or peritoneal fluid, while ST340 isolates (n = 5) were obtained from blood (n = 4) or urinary tract (n = 1) from infected patients. ST607 isolates (n = 6) were obtained from patients suffering from bloodstream infection (n = 3) or urinary tract infection (n = 3).
While the DRC lineages shared the same ST as K. pneumoniae isolates from several sub-Saharan African countries, their clustering and virulence gene content set them apart (Figure 1). Furthermore, 64.9% of DRC isolates shared the same ST as lineages found all over the world (e.g., ST14, ST15, ST39, ST48, ST340, and ST607). However, unlike the international clones which also belong to ST14 [37], ST15 [38], ST39 [39], ST48 [40], and ST340 [41,42], their DRC counterparts were susceptible to carbapenems and lacked carbapenemase gene.

3.4. Genetic Determinants of Virulence

3.4.1. Detection of Virulence Factors Genes

Among 128 virulence genes that were screened, 77 were found in at least one investigated isolate (Figure 1), with 27 of these virulence genes identified in each isolate. Table 3 summarizes data on ST, capsule typing, major virulence genes, and replicon typing. K. pneumoniae isolates harboured four distinct ybt: ybt9/conjugative integrative element (ICE) ICEKp3 (n = 6, among which 4 displayed a truncated ICE), ybt14/ICEKp15 (n = 12), ybt14/ICEKp12 (n = 2), ybt15/ ICEKp11 (n = 2), and ybt16/ICEKp12 (n = 4). The 37 K. pneumoniae isolates lacked the genes for colibactin (clb), salmochelin (iroBCDN), or aerobactin (iucABCD-iutA), as well as genes for hypervirulence (rmpA, rmpA2 genes, and peg-344) [43,44]. The virulence score was either of 0 or 1, reflecting the absence or presence of the ybt gene.

3.4.2. Replicon Typing

According to the FAB formula, the distribution of the replicon types among 33 of the 37 K. pneumoniae isolates was F13:A13:B− (n = 9), F13:A−:B− (n = 7), F−:A13:B− (n = 5), F2:A13:B− (n = 4), and F1:A13:B− (n = 3), while only one isolate had the replicon type F1:A−:B−, F2:A−:B−, F8:A13:B−, F9:A−:B−, or F12:A26:B−. The FIA26, a novel replicon, was reported for the first time in one isolate, whereas replicon types could not be characterized in four K. pneumoniae isolates. Apart from IncF replicons, no other incompatibility plasmid replicon types (i.e., IncA/C, IncH1, IncH2, IncI1, or IncN) were found in the K. pneumoniae isolates.

3.4.3. Identification of the Capsule Type

In the 37 K. pneumoniae isolates, five different O types were assigned: O1 (37.8%), O2a (13.5%), O2afg (8.1%), O3b (13.5%), and O4 (8.1%). No O type could be determined in 18.9% isolates. Only three different K types could be assigned to a total of five isolates: K2 in isolates CTMA_1655 (ST39) and CTMA_1682 (ST39); K14 in isolates CTMA_1656 and CTMA_1660 (ST37); and K62 in isolate CTMA_1684 (ST48). The remaining thirty-two isolates could not be assigned a K-type with certainty.

3.4.4. Detection of AMR Genes

AMR determinants included resistance genes and point mutations (Figure 2 and Figure 3, respectively). The four MDR ESBL-producing K. pneumoniae isolates that lacked the CTX-M-15 gene were CTMA_1654 (ST309), CTMA_1656 and CTMA_1660 (ST37), and CTMA_1688 (ST11). However, CTMA_1654 and CTMA_1656 carried blaSHV-11, while CTMA_1660 and CTMA_1688 harboured blaSHV12 and blaSHV, respectively.
In addition, other bla-genes associated with ESBL-phenotype were widespread in all MDR ESBL-producing K. pneumoniae isolates. These genes included blaSHV-1, blaSHV-28, blaSHV-62, blaSHV-110, and blaSHV-121. We observed a redundancy of ESBL-production associated genes, with several isolates harbouring blaCTX-M-15 (n = 33), blaTEM-1 (n = 31), and/or the above-cited blaSHV genes (n = 37). It is noteworthy that no isolates simultaneously carried multiple blaSHV or blaTEM. Other β-lactamases genes included blaOXA (n = 13), blaOXA-1 (n = 14), and blaSCO-1 (n = 6) [45].
All the MDR ESBL-producing K. pneumoniae isolates carried the fosA gene associated with resistance to fosfomycin. All DRC K. pneumoniae isolates carried at least one gene linked to resistance to aminoglycosides and/or quinolones gene (i.e., aac(3)-IId, aac(3)-IIe, aadA1, aadA16, aadA2, aph(3′)-Ia, aph(3′’)-Ib, aph(6)-Id, aac(6′)-Ib-cr, or aac(6′)-Ib-cr5), as well as to phenicols (catA1, catA2, and catB), sulfonamides (sul1 and sul2), tetracycline (tet(A), or tet(D)), and trimethoprim (dfrA1, dfrA12, dfrA14, dfrA15, dfrA25, and dfrA27). The two exceptions were the CTMA 1654 (ST309) and CTMA 1982 (ST2094) which lacked tetracycline resistance genes.
Plasmid-mediated AMR to quinolone was found in 26 K. pneumoniae isolates, with plasmid-mediated quinolone resistance (PMQR) genes qnrB, qnrB1, qnrB2, qnrB6, qnrS1 genes and plasmid-mediated efflux pumps genes oqxA, oqxB, oxqB5, oqxB19, oqxB21, and/or oqxB32.

3.4.5. Detection of AMR Point Mutations

In addition to AMR genes, AMR point mutations were identified (Figure 3). ST340 isolates (CTMA_1653, CTMA_1674, CTMA_1676, and 1678), and the ST11 isolate (CTMA_1688) carried the substitution S83I in the quinolone resistance-determining region (QRDR) of the gyrase A gene (gyrA), as well as the S80I substitution in the QRDR of the Deoxyribonucleic acid topoisomerase IV subunit A gene (parC). The two ST15 isolates CTMA_1657 and CTMA_1668 both had the S83F and D87A substitutions in gyrA, as well as the S80I substitution in the parC.
In ST2094 (CTMA_1682) and ST16 (CTMA_1689) isolates, the gyrA gene harboured the S83F and D87N substitutions, while parC had the E84K or S80I substitution. Likewise, ST874 isolates CTMA_1663 and CTMA_1667 both harboured the S83Y and D87A substi-tutions in gyrA as well as the S80I substitution in parC.
In the gyrA gene of five ST48 isolates, including CTMA_1650, CTMA_1661, CTMA_1662, CTMA_1666, and CTMA_1672, the S83Y substitution was the only mutation. The QRDR of parC from these isolates lacked any additional mutations.
Surprisingly, 9/37 (24,3%) MDR ESBL-producing K. pneumoniae isolates carried the colistin resistance-associated R256G substitution in the chromosomally encoded efflux pump pmrB gene. These isolates were typed ST340 (n = 5), ST37 (n = 2), ST11 (n = 1), and ST309 (n = 1). None of the K. pneumoniae isolates had any genes that were associated with reduced susceptibility to carbapenems.

4. Discussion

Being the cause of frequent outbreaks in hospitals, K. pneumoniae is becoming a significant global cause of severe infections. Despite reports of a high prevalence of MDR K. pneumoniae in a tertiary hospital in the eastern city of Bukavu in the DRC [16,17,22], neither this province nor the entire DRC have yet carried out molecular characterization of MDR ESBL-producing K. pneumoniae. We believe that this WGS study is the first to give insights on the genomic characteristics of MDR ESBL-producing K. pneumoniae isolates in this district of South-Kivu, eastern DRC. According to the WGS results, the MDR ESBL-producing K. pneumoniae isolates from clinical samples collected at the Bukavu hospital between 2013 and 2017 were found to have comparable ST to those commonly reported elsewhere.
The absence of carbapenemase genes, however, makes the current isolates significantly different from the global lineages, whose susceptibility to carbapenems is known to be decreased by these genetic determinants. A widespread use of carbapenems to treat severe ESBL-associated infection is likely too new in Bukavu to have seen the emergence of pandemic MDR ESBL-producing carbapenem-resistant K. pneumoniae (CRKP) lineages. This hypothesis is consistent with other data on MDR ESBL-producing K. pneumoniae in Ghana [46] and in southwestern Nigeria [47], where the administration of carbapenems was still restricted at the time of collection of isolates. In any case, the current results highlight the potential risk of a routine nontargeted administration of antimicrobial carbapenems, which is becoming more common in the province as a factor favouring the rapid emergence of carbapenem-resistant K. pneumoniae lineages.
A second difference between the current eastern DRC K. pneumoniae isolates is the absence of STs, which are commonly found in other MDR ESBL-producing K. pneumoniae global clonal lineages, notably ST101 [20,48], ST147 [20,49], ST258 [50], and ST307 [20,49,51,52].
When compared to several genomes from other sub-Saharan African countries, the higher content of virulence genes in the current eastern DRC K. pneumoniae isolates is a third difference (Figure 1). It is worth noting, however, that no hypervirulence gene was found in the eastern DRC isolates. This result is consistent with other data describing nonhypervirulent MDR ESBL-producing K. pneumoniae in hospital-acquired infections.
An intriguing finding is the presence of mutations associated with resistance to colistin in otherwise colistin-susceptible MDR ESBL-producing isolates. This observation was indeed made on isolates collected from patients whose treatment did not include colistin and in a province where there is no record of colistin administration. Regarding the use of colistin, these results should raise concerns about a potential rise of colistin-resistant MDR ESBL-producing K. pneumoniae, should this antibiotic be routinely introduced to treat infections.
Our study had several limitations. First, the relatively small number of isolates (37 out of 89 possible) is a source of potential bias, as we might have overlooked several phenotypic (antimicrobial susceptibility) and genetic features (phylogenomics, virulence factors and AMR genes) of several potential lineages of MDR ESBL-producing K. pneumoniae in this tertiary care hospital. Secondly, several sequences including those on chromosome and genetic mobile elements can be missing from the draft genomes generated by Illumina short reads. Their use in subsequent genomic analyses can be potentially hampered by this incompleteness [53]. This is evidenced by the failure to assign any replicon to isolates carrying plasmid-mediated AMR genes (i.e., isolates CTMA_1659; CTMA_1664, and CTMA_1667).
In conclusion, our study, despite its limitations, highlights the high genetic variability of MDR ESBL-producing K. pneumoniae in this easternmost province of DRC, calls for additional research across the whole country to determine the prevalence of these MDR ESBL-producing K. pneumoniae isolates, and suggests characterizing AMR genetic determinants, as well as virulence factor genes, that could account for their spread in the country and beyond. In practice, these results call for caution when administering colistin and carbapenems as well as parallel monitoring of the genomic and phenotypic evolution of AMR in DRC.

Author Contributions

Conceptualization: L.M.I., J.-L.G. Methodology: L.M.I., J.A., B.B., J.-F.D., M.B. Validation: L.M.I., J.A., B.B., J.-F.D. Formal analysis: L.M.I., J.A., M.B. Writing—original draft preparation: L.M.I., J.A. Writing—review and editing: L.M.I., J.A., J.-L.G. Supervision: J.-L.G. Project administration: L.M.I., J.-L.G. Funding Acquisition: L.M.I., J.-L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Belgian Cooperation Agency of the ARES (Académie de Recherche et d’Enseignement Supérieur) [grant COOP-CONV-20-022]. M.B. was founded by European Union’s Horizon 2020 research and innovation program under grant agreement No 883285. The material presented and views expressed here are the responsibility of the author(s) only. The EU Commission takes no responsibility for any use made of the information set out.

Data Availability Statement

Publicly available datasets were analysed in this study. Raw sequencing data from all K. pneumoniae isolates were submitted to the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena) and are available under accession number PRJEB56212 numbers. The data used and/or analysed during the current study are available from the corresponding author upon reasonable request. The DNA sequence of the novel FIA replicon was submitted to the curator of the pMLST database (https://pubmlst.org/plasmid/; submitted on 26 July 2022) for ST assignment.

Acknowledgments

We acknowledge the assistance of Raphaël B. Chirimwami (Université Catholique de Bukavu), and Michèle Bouyer (Defence Laboratories Department) for providing technical assistance in antimicrobial susceptibility assays on K. pneumoniae isolates.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Tseng, W.P.; Chen, Y.C.; Chen, S.Y.; Chen, S.Y.; Chang, S.C. Risk for subsequent infection and mortality after hospitalization among patients with multidrug-resistant gram-negative bacteria colonization or infection. Antimicrob. Resist. Infect. Control 2018, 7, 93. [Google Scholar] [CrossRef] [Green Version]
  2. Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson, M.; Monnet, D.L.; Pulcini, C.; Kahlmeter, G.; Kluytmans, J.; Carmeli, Y.; et al. Discovery, research, and development of new antibiotics: The WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 2018, 18, 318–327. [Google Scholar] [CrossRef] [PubMed]
  3. Saravanan, M.; Ramachandran, B.; Barabadi, H. The prevalence and drug resistance pattern of extended spectrum β-lactamases (ESBLs) producing Enterobacteriaceae in Africa. Microb. Pathog. 2018, 114, 180–192. [Google Scholar] [CrossRef] [PubMed]
  4. Casellas, J.M. Antibacterial drug resistance in Latin America: Consequences for infectious disease control. Rev. Panam Salud Publica 2011, 30, 519–528. [Google Scholar] [PubMed]
  5. Paterson, D.L.; Bonomo, R.A. Extended-spectrum β-lactamases: A clinical update. Clin. Microbiol. Rev. 2005, 18, 657–686. [Google Scholar] [CrossRef] [Green Version]
  6. Pitout, J.D.D. Enterobacteriaceae that produce extended-spectrum β-lactamases and AmpC β-lactamases in the community: The tip of the iceberg? Curr. Pharm. Des. 2013, 19, 257–263. [Google Scholar] [CrossRef]
  7. Peirano, G.; Pitout, J.D.D. Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae: Update on Molecular Epidemiology and Treatment Options. Drugs 2019, 79, 1529–1541. [Google Scholar] [CrossRef]
  8. Aibinu, I.; Odugbemi, T.; Koenig, W.; Ghebremedhin, B. Sequence type ST131 and ST10 complex (ST617) predominant among CTX-M-15-producing Escherichia coli isolates from Nigeria. Clin. Microbiol. Infect. 2012, 18, E49–E51. [Google Scholar] [CrossRef] [Green Version]
  9. Irenge, L.M.; Ambroise, J.; Bearzatto, B.; Durant, J.F.; Chirimwami, R.B.; Gala, J.L. Whole-genome sequences of multi-drug-resistant Escherichia coli in South-Kivu Province, Democratic Republic of Congo: Characterization of phylogenomic changes, virulence and resistance genes. BMC Infect. Dis. 2019, 19, 137. [Google Scholar] [CrossRef] [Green Version]
  10. Martischang, R.; Francois, P.; Cherkaoui, A.; Gaia, N.; Renzi, G.; Agostinho, A.; Perez, M.; Graf, C.E.; Harbarth, S. Epidemi-ology of ESBL-producing Escherichia coli from repeated prevalence studies over 11 years in a long-term-care facility. Antimicrob. Resist. Infect. Control 2021, 10, 148. [Google Scholar] [CrossRef]
  11. Eger, E.; Heiden, S.E.; Korolew, K.; Bayingana, C.; Ndoli, J.M.; Sendegeya, A.; Gahutu, J.B.; Kurz, M.S.E.; Mockenhaupt, F.P.; Muller, J.; et al. Circulation of Extended-Spectrum β-Lactamase-Producing Escherichia coli of Pandemic Sequence Types 131, 648, and 410 Among Hospitalized Patients, Caregivers, and the Community in Rwanda. Front. Microbiol. 2021, 12, 662575. [Google Scholar] [CrossRef]
  12. Tegha, G.; Ciccone, E.J.; Krysiak, R.; Kaphatika, J.; Chikaonda, T.; Ndhlovu, I.; van Duin, D.; Hoffman, I.; Juliano, J.J.; Wang, J. Genomic epidemiology of Escherichia coli isolates from a tertiary referral center in Lilongwe, Malawi. Microb. Genom. 2021, 7. [Google Scholar] [CrossRef] [PubMed]
  13. Seni, J.; Peirano, G.; Mshana, S.E.; Pitout, J.D.D.; DeVinney, R. The importance of Escherichia coli clonal complex 10 and ST131 among Tanzanian patients on antimicrobial resistance surveillance programs. Eur. J. Clin. Microbiol. Infect. Dis. 2021. [Google Scholar] [CrossRef] [PubMed]
  14. Bastidas-Caldes, C.; Romero-Alvarez, D.; Valdez-Velez, V.; Morales, R.D.; Montalvo-Hernandez, A.; Gomes-Dias, C.; Calopina, M. Extended-Spectrum β-Lactamases Producing Escherichia coli in South America: A Systematic Review with a One Health Perspective. Infect. Drug Resist. 2022, 15, 5759–5779. [Google Scholar] [CrossRef] [PubMed]
  15. Onduru, O.G.; Mkakosya, R.S.; Aboud, S.; Rumisha, S.F. Genetic Determinants of Resistance among ESBL-Producing Enterobacteriaceae in Community and Hospital Settings in East, Central, and Southern Africa: A Systematic Review and Meta-Analysis of Prevalence. Can. J. Infect. Dis. Med. Microbiol. 2021, 2021, 5153237. [Google Scholar] [CrossRef]
  16. Irenge, L.M.; Kabego, L.; Vandenberg, O.; Chirimwami, R.B.; Gala, J.L. Antimicrobial resistance in urinary isolates from inpatients and outpatients at a tertiary care hospital in South-Kivu Province (Democratic Republic of Congo). BMC Res. Notes 2014, 7, 374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Irenge, L.M.; Kabego, L.; Kinunu, F.B.; Itongwa, M.; Mitangala, P.N.; Gala, J.L.; Chirimwami, R.B. Antimicrobial resistance of bacteria isolated from patients with bloodstream infections at a tertiary care hospital in the Democratic Republic of the Congo. S. Afr. Med. J. 2015, 105, 752–755. [Google Scholar] [CrossRef] [Green Version]
  18. Bitew, A.; Tsige, E. High Prevalence of Multidrug-Resistant and Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae: A Cross-Sectional Study at Arsho Advanced Medical Laboratory, Addis Ababa, Ethiopia. J. Trop. Med. 2020, 2020, 6167234. [Google Scholar] [CrossRef]
  19. Moglad, E.H. Antibiotics Profile, Prevalence of Extended-Spectrum β-Lactamase (ESBL), and Multidrug-Resistant Enterobacteriaceae from Different Clinical Samples in Khartoum State, Sudan. Int. J. Microbiol. 2020, 2020, 8898430. [Google Scholar] [CrossRef]
  20. Osman, E.A.; Yokoyama, M.; Altayb, H.N.; Cantillon, D.; Wille, J.; Seifert, H.; Higgins, P.G.; Al Hassan, L. Klebsiella pneumonia in Sudan: Multidrug Resistance, Polyclonal Dissemination, and Virulence. Antibiotics 2023, 12, 233. [Google Scholar] [CrossRef]
  21. Muraya, A.; Kyany’a, C.; Kiyaga, S.; Smith, H.J.; Kibet, C.; Martin, M.J.; Kimani, J.; Musila, L. Antimicrobial Resistance and Virulence Characteristics of Klebsiella pneumoniae Isolates in Kenya by Whole-Genome Sequencing. Pathogens 2022, 11, 545. [Google Scholar] [CrossRef] [PubMed]
  22. Wyres, K.L.; Holt, K.E. Klebsiella pneumoniae Population Genomics and Antimicrobial-Resistant Clones. Trends Microbiol. 2016, 24, 944–956. [Google Scholar] [CrossRef] [Green Version]
  23. Mulinganya, G.M.; Claeys, M.; Balolebwami, S.Z.; Bamuleke, B.A.; Mongane, J.I.; Boelens, J.; Delanghe, J.; De Vos, D.; Kam-bale, R.M.; Maheshe, G.B.; et al. Etiology of Early-Onset Neonatal Sepsis and Antibiotic Resistance in Bukavu, Democratic Republic of the Congo. Clin. Infect. Dis. 2021, 73, e976–e980. [Google Scholar] [CrossRef] [PubMed]
  24. Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data. v.0.11.9. 2010. Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 27 January 2023).
  25. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Gurevich, A.; Saveliev, V.; Vyahhi, N.; Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 2013, 29, 1072–1075. [Google Scholar] [CrossRef] [Green Version]
  28. Seppey, M.; Manni, M.; Zdobnov, E.M. BUSCO: Assessing Genome Assembly and Annotation Completeness. Methods Mol. Biol. 2019, 1962, 227–245. [Google Scholar] [CrossRef]
  29. Wattam, A.R.; Abraham, D.; Dalay, O.; Disz, T.L.; Driscoll, T.; Gabbard, J.L.; Gillespie, J.J.; Gough, R.; Hix, D.; Kenyon, R.; et al. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res. 2014, 42, D581–D591. [Google Scholar] [CrossRef] [Green Version]
  30. Gardner, S.N.; Hall, B.G. When whole-genome alignments just won’t work: kSNP v2 software for alignment-free SNP discovery and phylogenetics of hundreds of microbial genomes. PLoS One 2013, 8, e81760. [Google Scholar] [CrossRef]
  31. Yu, G. Using ggtree to Visualize Data on Tree-Like Structures. Curr. Protoc. Bioinform. 2020, 69, e96. [Google Scholar] [CrossRef]
  32. Chen, L.; Yang, J.; Yu, J.; Yao, Z.; Sun, L.; Shen, Y.; Jin, Q. VFDB: A reference database for bacterial virulence factors. Nucleic Acids Res. 2005, 33, D325–D328. [Google Scholar] [CrossRef] [Green Version]
  33. Lam, M.M.C.; Wick, R.R.; Watts, S.C.; Cerdeira, L.T.; Wyres, K.L.; Holt, K.E. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat. Commun. 2021, 12, 4188. [Google Scholar] [CrossRef] [PubMed]
  34. Wyres, K.L.; Wick, R.R.; Gorrie, C.; Jenney, A.; Follador, R.; Thomson, N.R.; Holt, K.E. Identification of Klebsiella capsule synthesis loci from whole genome data. Microb. Genom. 2016, 2, e000102. [Google Scholar] [CrossRef] [PubMed]
  35. Feldgarden, M.; Brover, V.; Gonzalez-Escalona, N.; Frye, J.G.; Haendiges, J.; Haft, D.H.; Hoffmann, M.; Pettengill, J.B.; Prasad, A.B.; Tillman, G.E.; et al. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci. Rep. 2021, 11, 12728. [Google Scholar] [CrossRef] [PubMed]
  36. Villa, L.; Garcia-Fernandez, A.; Fortini, D.; Carattoli, A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J. Antimicrob. Chemother. 2010, 65, 2518–2529. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Moubareck, C.A.; Mouftah, S.F.; Pal, T.; Ghazawi, A.; Halat, D.H.; Nabi, A.; AlSharhan, M.A.; AlDeesi, Z.O.; Peters, C.C.; Celiloglu, H.; et al. Clonal emergence of Klebsiella pneumoniae ST14 co-producing OXA-48-type and NDM carbapenemases with high rate of colistin resistance in Dubai, United Arab Emirates. Int. J. Antimicrob. Agents 2018, 52, 90–95. [Google Scholar] [CrossRef] [PubMed]
  38. Martins, W.; Nicolas, M.F.; Yu, Y.; Li, M.; Dantas, P.; Sands, K.; Portal, E.; Almeida, L.G.P.; Vasconcelos, A.T.R.; Medeiros, E.A.; et al. Clinical and Molecular Description of a High-Copy IncQ1 KPC-2 Plasmid Harbored by the International ST15 Klebsiella pneumoniae Clone. mSphere 2020, 5, e00756-20. [Google Scholar] [CrossRef]
  39. Karampatakis, T.; Zarras, C.; Pappa, S.; Vagdatli, E.; Iosifidis, E.; Roilides, E.; Papa, A. Emergence of ST39 carbapenem-resistant Klebsiella pneumoniae producing VIM-1 and KPC-2. Microb. Pathog. 2022, 162, 105373. [Google Scholar] [CrossRef] [PubMed]
  40. Gu, B.; Bi, R.; Cao, X.; Qian, H.; Hu, R.; Ma, P. Clonal dissemination of KPC-2-producing Klebsiella pneumoniae ST11 and ST48 clone among multiple departments in a tertiary teaching hospital in Jiangsu Province, China. Ann. Transl. Med. 2019, 7, 716. [Google Scholar] [CrossRef]
  41. Tolentino, F.M.; Bueno, M.F.C.; Franscisco, G.R.; Barcelos, D.D.P.; Lobo, S.M.; Tomaz, F.; da Silva, N.S.; de Andrade, L.N.; Casella, T.; Darini, A.; et al. Endemicity of the High-Risk Clone Klebsiella pneumoniae ST340 Coproducing QnrB, CTX-M-15, and KPC-2 in a Brazilian Hospital. Microb. Drug. Resist. 2019, 25, 528–537. [Google Scholar] [CrossRef]
  42. Peirano, G.; Bradford, P.A.; Kazmierczak, K.M.; Chen, L.; Kreiswirth, B.N.; Pitout, J.D. Importance of Clonal Complex 258 and IncF(K2-like) Plasmids among a Global Collection of Klebsiella pneumoniae with bla(KPC). Antimicrob. Agents Chemother. 2017, 61, e02610–e02616. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Russo, T.A.; Marr, C.M. Hypervirulent Klebsiella pneumoniae. Clin. Microbiol. Rev. 2019, 32, e00001-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Remya, P.A.; Shanthi, M.; Sekar, U. Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae. Indian J. Med. Microbiol. 2019, 37, 210–218. [Google Scholar] [CrossRef] [PubMed]
  45. Poirel, L.; Corvec, S.; Rapoport, M.; Mugnier, P.; Petroni, A.; Pasteran, F.; Faccone, D.; Galas, M.; Drugeon, H.; Cattoir, V.; et al. Identification of the novel narrow-spectrum β-lactamase SCO-1 in Acinetobacter spp. from Argentina. Antimicrob. Agents Chemother. 2007, 51, 2179–2184. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Agyepong, N.; Govinden, U.; Owusu-Ofori, A.; Amoako, D.G.; Allam, M.; Janice, J.; Pedersen, T.; Sundsfjord, A.; Essack, S. Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital. Int. J. Infect. Dis. 2019, 85, 117–123. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Afolayan, A.O.; Oaikhena, A.O.; Aboderin, A.O.; Olabisi, O.F.; Amupitan, A.A.; Abiri, O.V.; Ogunleye, V.O.; Odih, E.E.; Adeyemo, A.T.; Adeyemo, A.T.; et al. Clones and Clusters of Antimicrobial-Resistant Klebsiella From Southwestern Nigeria. Clin. Infect. Dis. 2021, 73, S308–S315. [Google Scholar] [CrossRef]
  48. Can, F.; Menekse, S.; Ispir, P.; Atac, N.; Albayrak, O.; Demir, T.; Karaaslan, D.C.; Karahan, S.N.; Kapmaz, M.; Kurt Azap, O.; et al. Impact of the ST101 clone on fatality among patients with colistin-resistant Klebsiella pneumoniae infection. J. Antimicrob. Chemother. 2018, 73, 1235–1241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Peirano, G.; Chen, L.; Kreiswirth, B.N.; Pitout, J.D.D. Emerging Antimicrobial-Resistant High-Risk Klebsiella pneumoniae Clones ST307 and ST147. Antimicrob. Agents Chemother. 2020, 64, e01148-20. [Google Scholar] [CrossRef]
  50. Marsh, J.W.; Mustapha, M.M.; Griffith, M.P.; Evans, D.R.; Ezeonwuka, C.; Pasculle, A.W.; Shutt, K.A.; Sundermann, A.; Ayres, A.M.; Shields, R.K.; et al. Evolution of Outbreak-Causing Carbapenem-Resistant Klebsiella pneumoniae ST258 at a Tertiary Care Hospital over 8 Years. mBio 2019, 10, e01945-19. [Google Scholar] [CrossRef] [Green Version]
  51. Villa, L.; Feudi, C.; Fortini, D.; Brisse, S.; Passet, V.; Bonura, C.; Endimiani, A.; Mammina, C.; Ocampo, A.M.; Jimenez, J.N.; et al. Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone. Microb. Genom. 2017, 3, e000110. [Google Scholar] [CrossRef]
  52. Loconsole, D.; Accogli, M.; De Robertis, A.L.; Capozzi, L.; Bianco, A.; Morea, A.; Mallamaci, R.; Quarto, M.; Parisi, A.; Chironna, M. Emerging high-risk ST101 and ST307 carbapenem-resistant Klebsiella pneumoniae clones from bloodstream infections in Southern Italy. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 24. [Google Scholar] [CrossRef] [PubMed]
  53. Peona, V.; Weissensteiner, M.H.; Suh, A. How complete are “complete” genome assemblies? An avian perspective. Mol. Ecol. Resour. 2018, 18, 1188–1195. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Microorganisms 11 00525 g001 550
Figure 1. Phylogenetic tree of MDR ESBL-producing K. pneumoniae isolates from the DRC and other countries. The heatmap of K. pneumoniae-related virulence genes from the Virulence Factor Database (VFDB) is shown on the right side of the figure. The kSNP3 parsimony tree is based on single nucleotide polymorphisms (SNPs) within the core genomes of K. pneumoniae isolates from the DRC (in black) as well as isolates from sub-Saharan countries such as Nigeria, Tanzania, and South Africa (in grey). The heatmap’s red cells represent highly (>80%) covered genes, while the orange and yellow cells represent partially (50—80%) covered and uncovered (<50%) genes, respectively. Several genes, including entD and vgrG, have large deletions. acrA, acrB, dotU-tssL, entA, entB, entC, fepA, fepB, fepC, fepD, fepG, fes, hcp-tssD, iutA1, mrkC are examples of ubiquitous virulence genes.
Figure 1. Phylogenetic tree of MDR ESBL-producing K. pneumoniae isolates from the DRC and other countries. The heatmap of K. pneumoniae-related virulence genes from the Virulence Factor Database (VFDB) is shown on the right side of the figure. The kSNP3 parsimony tree is based on single nucleotide polymorphisms (SNPs) within the core genomes of K. pneumoniae isolates from the DRC (in black) as well as isolates from sub-Saharan countries such as Nigeria, Tanzania, and South Africa (in grey). The heatmap’s red cells represent highly (>80%) covered genes, while the orange and yellow cells represent partially (50—80%) covered and uncovered (<50%) genes, respectively. Several genes, including entD and vgrG, have large deletions. acrA, acrB, dotU-tssL, entA, entB, entC, fepA, fepB, fepC, fepD, fepG, fes, hcp-tssD, iutA1, mrkC are examples of ubiquitous virulence genes.
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Figure 2. Heatmap of the genes involved in Antimicrobial Resistance (AMR) and identified with the AMRFinder tool. Black cells indicate the presence of the corresponding AMR gene.
Figure 2. Heatmap of the genes involved in Antimicrobial Resistance (AMR) and identified with the AMRFinder tool. Black cells indicate the presence of the corresponding AMR gene.
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Figure 3. Heatmap of antimicrobial resistance (AMR) point mutations identified with the AMRFinder tool. Black cells indicate the presence of the corresponding SNP determinant.
Figure 3. Heatmap of antimicrobial resistance (AMR) point mutations identified with the AMRFinder tool. Black cells indicate the presence of the corresponding SNP determinant.
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Table
Table 1. Antimicrobial susceptibility profiles of ESBL-producing K. pneumoniae isolates (n = 37) from the DRC. MIC values (mg/L) for tested antimicrobial agents.
Table 1. Antimicrobial susceptibility profiles of ESBL-producing K. pneumoniae isolates (n = 37) from the DRC. MIC values (mg/L) for tested antimicrobial agents.
IsolateTissueSTAMPAMXAMCFEPCTXCAZCROIPMMEROCIPAKNGENTTETCOLNITRO
CTMA-1678Bloodstream340>256>256>25648>256>256>320.0940.75>321296>2560.25>512
CTMA-1674Bloodstream340>256>256>25664>256>256>320.0940.094>326>256>2560.25>512
CTMA-1676Bloodstream340>256>256>25664>256>256>320.0940.047>32864>2560.25>512
CTMA-1653Bloodstream340>256>256>25632>256>256>320.50.47>32864>2560.25>512
CTMA-1690UTI340>256>256>25664>256192>320.190.094>328128>256<0.25>512
CTMA-1668UTI15>256>256>25648>256>256>320.250.032>32696>2560.2548
CTMA-1660UTI37>256>256160.251212>320.1250.023124>256>256<0.253
CTMA-1656UTI37>256>256>2560.752424>320.190.23132>256>2560.256
CTMA-1654UTI309>256>256>25612>256>256>320.1250.032>3224>256>2560.2564
CTMA-1689UTI16>256>256>25648>256>256>320.250.002>32896>2560.25>512
CTMA-1682UTI2094>256>256>25664>256>256>320.0230.125>32124840.532
CTMA-1677Bloodstream1777>256>256>256126416>320.0940.0641.5396>2560.25>512
CTMA-1673Bloodstream1777>256>2561212>256>256>320.250.0471.5396>2560.25128
CTMA-1665Peritoneal fluid502>256>256>25624>256>256>320.0020.23424128>256<0.2512
CTMA-1680Bloodstream39>256>256>2562496>256>320.1250.032348>256>2560.2596
CTMA-1679Bloodstream39>256>256>25624>256>256>320.1250.064248>256>2560.2532
CTMA-1681Bloodstream39>256>256>25632>25632>320.1250.032264>256>2560.596
CTMA-1655UTI39>256>256>25632>256>256>320.190.32224>256>2560.256
CTMA-1664UTI967>256>25688>25624>320.0640.471.5824>2560.2564
CTMA-1659UTI337>256>256824>25616>320.1250.050.7520.75>2560.2524
CTMA-1661Soft tissue48>256>256>25616>256>256>320.0940.0236632>256<0.25128
CTMA-1666Bloodstream48>256>256648>256>256>320.0230.1251.582464<0.2596
CTMA-1672Bloodstream48>256>2561.516>256>256>320.190.047>321696>2560.25>512
CTMA-1684Bloodstream48>256>256>25616>256>256>320.0940.04781232160.5128
CTMA-1650Bloodstream48>256>256>25616>256>256>320.0940.023>321696>2560.25>512
CTMA-1662Soft tissue48>256>2566424>256>256>320.0940.0236632>256<0.25128
CTMA-1667Bloodstream874>256>256>2561.5>256>256>320.0470.0940.5380.094>2560.2516
CTMA-1663Bloodstream874>256>2564824>256>256>320.50.047>32896>2560.25192
CTMA-1683Bloodstream607>256>256>25616>256>256>320.2560.12524128>2560.25>512
CTMA-1687UTI607>256>256>256>256>256>256>320.0940.0641.0003>256>2560.5>512
CTMA-1686UTI607>256>256>256>256>256>256>320.190.0230.09461921.50.5>512
CTMA-1671Bloodstream607>256>256>2561.5>256>256>320.0940.047>32.00012128>2560.25>512
CTMA-1675Bloodstream607>256>256>25648>256>256>320.1250.0470.1254>256>2560.25192
CTMA-1652UTI607>256>256>256>256>256>256>320.1250.470.1254>256>2560.25>512
CTMA-1685UTI14>256>256>2566>256>256>320.190.0231.56961280.5>512
CTMA-1657UTI15>256>256>25616>256>256>320.1250.06>32864>2560.2564
CTMA-1688Bloodstream11>256>256>25648>256>256>320.2560.0231.50066420.2548
Abbreviation: ST: Sequence Type; AKN: Amikacin; GENT: Gentamicin, AMP: Ampicillin; AMX: Amoxicillin; FEP: Cefepime; IMP: Imipenem; MERO: Meropenem; AMC: Amoxicillin/Clavulanic Acid; CAZ: Ceftazidime; CTX: Cefotaxime; CRO: Ceftriaxone; CIP: Ciprofloxacin; NITRO: Nitrofurantoin; COL: Colistin. R: Resistant, S: Susceptible, UTI: Urinary tract infection.
Table
Table 2. AMR percentages of ESBL-producing K. Pneumoniae isolates according to CLSI/EUCAST guidelines.
Table 2. AMR percentages of ESBL-producing K. Pneumoniae isolates according to CLSI/EUCAST guidelines.
AntibioticsResistance (R)
n = 37 (%)		Susceptible (S)
n = 37 (%)		Area of Technical Uncertainty (ATU)
n = 37 (%)
Ampicillin37 (100)0 (0)0 (0)
Amoxicillin37 (100)0 (0)0 (0)
Amoxicillin-clavulanate35 (94.6)2 (5.4)0 (0)
Cefepime34 (91.9)3 (8.1)0 (0)
Cefotaxime37 (100)0 (0)0 (0)
Ceftazidime37 (100)0 (0)0 (0)
Ceftriaxone37 (100)0 (0)0 (0)
Imipenem0 (0)37 (100)0 (0)
Meropenem0 (0)37 (100)0 (0)
Ciprofloxacin35 (94.6)1 (2.7)1 (2.7)
Amikacin15 (40.5)22 (59.5)0 (0)
Gentamicin35 (94.6)2 (5.4)0 (0)
Tetracycline34 (91.9)3 (8.1)0 (0)
Colistin0 (0)37 (100)0 (0)
Nitrofurantoin24 (64.9)13 (35.1)0 (0)
Table
Table 3. MLST sequence type (ST), K and O and their respective K (capsule) and O antigen (LPS) type, as predicted by Kleborate.
Table 3. MLST sequence type (ST), K and O and their respective K (capsule) and O antigen (LPS) type, as predicted by Kleborate.
Year of IsolationStrainOriginSTReplicon STK_TypeO TypeYersiniabactin(Hyper) Virulence GenesVirulence Score
2014CTMA_1678BloodstreamST340F1:A−:B− unknown (KL107)unknown (O4)ybt 9; ICEKp3 (truncated)-1
2014CTMA_1674BloodstreamST340F1:A13:B−unknown (K15)O4ybt 9; ICEKp3 (truncated)-1
2014CTMA_1676BloodstreamST340F1:A13:B−unknown (K15)O4ybt 9; ICEKp3 (truncated)-1
2014CTMA_1653BloodstreamST340F1:A13:B− unknown (KL107)unknown (O4)ybt 9; ICEKp3 (truncated)-1
2013CTMA_1690UTIST340F−:A13:B−unknown (K15)O4ybt 14; ICEKp5-1
2013CTMA_1688UTIST11F13:A13:B−unknown (KL105)O2afgybt 9; ICEKp3-1
2014CTMA_1660UTIST37F−:A13:B−K14O3bybt 15; ICEKp11-1
2014CTMA_1656UTIST37F−:A13:B−K14O3bybt 15; ICEKp11-1
2014CTMA_1654UTIST309NDunknown (K42)unknown (O4)--0
2013CTMA_1689UTIST16F12:A26:B−unknown (K51)O3bybt 9; ICEKp3-1
2016CTMA_1682UTIST2094F−:A13:B−unknown (KL107)unknown (OL101)--0
2014CTMA_1677BloodstreamST1777F13:A13:B−unknown (KL111)O3b--0
2014CTMA_1673BloodstreamST1777F13:A13:B−unknown (KL111)O3b--0
2015CTMA_1665Soft tissueST502F13:A−:B−unknown (K15)unknown (O4)--0
2015CTMA_1680BloodstreamST39F2:A13:B−unknown (K2)O2aybt 16; ICEKp12-1
2015CTMA_1679BloodstreamST39F2:A13:B−unknown (K2)O1ybt 16; ICEKp12-1
2015CTMA_1681BloodstreamST39F2:A13:B−unknown (K2)O1ybt 16; ICEKp12-1
2015CTMA_1655UTIST39F2:A13:B−K2O1ybt 16; ICEKp12-1
2014CTMA_1664UTIST967F2:A−:B−unknown (K18)O2afg--0
2014CTMA_1659UTIST337NDunknown (KL109)O2afg--0
2015CTMA_1661Peritoneal fluidST48F13:A−:B−unknown (K62)O1ybt 14; ICEKp5-1
2014CTMA_1666BloodstreamST48F13:A−:B−unknown (K62)O2aybt 14; ICEKp5-1
2015CTMA_1672BloodstreamST48F13:A−:B−unknown (K62)unknown (O2a)ybt 14; ICEKp5-1
2016CTMA_1684BloodstreamST48F13:A−:B−unknown (K62)O1ybt 14; ICEKp5-1
2014CTMA_1650BloodstreamST48F13:A−:B−K62O1ybt 14; ICEKp5-1
2015CTMA_1662Peritoneal fluidST48F13:A−:B−unknown (K62)O1ybt 14; ICEKp5-1
2014CTMA_1667BloodstreamST874NDunknown (K45)O1ybt 14; ICEKp12-1
2015CTMA_1663BloodstreamST874NDunknown (K45)unknown (O1)ybt 14; ICEKp12-1
2017CTMA_1683BloodstreamST607F13:A13:B−unknown (K25)O1ybt 14; ICEKp5-1
2014CTMA_1687UTIST607F13:A13:B−unknown (K25)O1--0
2017CTMA_1686UTIST607F13:A13:B−unknown (K25)O1ybt 14; ICEKp5-1
2014CTMA_1671BloodstreamST607F13:A13:B−unknown (K25)O2aybt 14; ICEKp5-1
2017CTMA_1675BloodstreamST607F13:A13:B−unknown (K25)O1ybt 14; ICEKp5-1
2015CTMA_1652UTIST607F13:A13:B−unknown (K25)O2aybt 14; ICEKp5-1
2013CTMA_1685UTIST14F9:A−:B−K2O1- 0
2014CTMA_1657UTIST15F8:A13:B−unknown (KL112)O1--0
2013CTMA_1668BloodstreamST15F−:A13:B−unknown (K48)O2a--0
Abbreviation: UTI: Urinary tract infection. The Yersiniabactin column represents ybt genes alleles and their respective ICEKp. The virulence genes (colibactin (clb), aerobactin (iucABCD-iutA), salmochelin (iroBCDN)), as well as hypervirulence genes (rmpA, rmpA2 genes, and peg-344) were not identified in the 37 isolates.
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MDPI and ACS Style

Irenge, L.M.; Ambroise, J.; Bearzatto, B.; Durant, J.-F.; Bonjean, M.; Gala, J.-L. Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo. Microorganisms 2023, 11, 525. https://doi.org/10.3390/microorganisms11020525

AMA Style

Irenge LM, Ambroise J, Bearzatto B, Durant J-F, Bonjean M, Gala J-L. Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo. Microorganisms. 2023; 11(2):525. https://doi.org/10.3390/microorganisms11020525

Chicago/Turabian Style

Irenge, Leonid M., Jérôme Ambroise, Bertrand Bearzatto, Jean-François Durant, Maxime Bonjean, and Jean-Luc Gala. 2023. "Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo" Microorganisms 11, no. 2: 525. https://doi.org/10.3390/microorganisms11020525

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